A molecular description of synaptogenesis remains a key research goal in understanding the development and function of the nervous system. By characterizing C. elegans genes that function in synapse formation, our work has contributed to the discovery of several signaling pathways instructing different aspects of presynaptic differentiation. Central to this application is the rpm-1 gene (for regulator of presynaptic morphology). Loss of function in rpm-1 causes disorganized presynaptic architecture and disrupts axonal patterning in a neuron-type specific manner. RPM-1 is a member of the conserved PHR protein family that includes mammalian Pam and Phr1, and Drosophila Highwire. PHR proteins are large molecules containing multiple functional domains, including an RCC1 guanine exchange factor homology domain and a Ring-finger E3 ubiquitin ligase domain. During the current funding period, we demonstrated that RPM-1 functions as an E3 ubiquitin ligase for the conserved MAP kinase kinase kinase DLK-1. DLK-1 activates two downstream kinases, a MAPKK MKK-4 and a p38 MAPK PMK-3. Down-regulation of this MAP kinase cascade by RPM-1 is essential for normal synapse formation. In parallel to the genetic approaches, we used biochemical methods to identify RPM-1 associated proteins, and discovered that RPM-1 positively regulates late endo-lysosomal trafficking via the RabGEF GLO-4. The overall goals of the present application are to define the targets of the RPM-1/MAPK cascade, and to understand how the cascade is regulated. We have identified two new genes, mak-2, the C. elegans ortholog of MAP kinase activated kinase 2, and uev-3, a protein containing an inactive ubiquitin conjugating enzyme domain. Loss of function in either gene behaves in a manner similar to that of inactivating the MAPK cascade. Our preliminary studies suggest that MAK-2 and UEV-3 act downstream of DLK-1 and MKK-4. In aim-1, we will examine the regulation of MAK-2 by the MAP kinases and identify the targets of MAK-2. In aim- 2, we will analyze the interaction between UEV-3 and the MAP kinases. In aim-3, we will explore a novel pathway in synapse development. Loss of function in individual synaptogenic pathways has mild effects on synaptic development and function, indicating a high degree of functional redundancy in synaptic signaling. Using genetic modifier screens to search for other synapse development genes, we have identified the gene sydn-1 (for syd enhancer), which appears to define a nuclear pathway that may regulate axonal pruning in a synaptogenesis dependent manner. We will investigate the cellular and molecular functions of SYDN-1 and its candidate interacting genes. Successful completion of our aims will elucidate how the PHR/MAPK pathway functions. Regulation of synapse stability is one of the major pathogenesis events associated with dementia and ageing. This study will contribute to the understanding of the basic mechanisms that build and maintain synapses, and may also provide insights into the pathogenesis of synapse dysfunction. PUBLIC HEALTH RELEVANCE: This application investigates the signal transduction pathway of a p38 MAP kinase in synapse development. It will determine the molecular, biochemical, and cellular interactions of a MAPKAP protein and a UEV-domain containing protein with the MAP kinase cascade. It will provide insights into the basic mechanisms controlling synapse stability and into the pathogenesis process underlying synapse dysfunction in diseases.